The invention relates to novel human phosphatidylserine synthase-like nucleic acid sequences and proteins. Also provided are vectors, host cells, and recombinant methods for making and using the novel molecules.
The membranes of eukaryotic cells contain not only large amounts of cholesterol but a variety of phospholipids. For example, the major phospholipids in the human erythrocyte include phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin. The four major phospholipids in the yeast Saccharomyces cerevisiae are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) (Poole et al. (1986) J. of Bacteriology 168(2):668-672). PS accounts for 4 to 8% of the total membrane phospholipids in S. cerevisiae and is important to overall lipid metabolism (Atkinson et al. (1980) J. Biol. Chem. 255: 6653-6661). PS is the normal precursor to PE and PC. The synthesis of phospholipids in eukaryotic cells involves both cytoplasmic and membrane-associated enzymes, a number of which are coordinately regulated (Henry et al., (1984) Annu. Rev. Genet. 18:207-231).
Phosphatidylserine (PS) is an essential phospholipid for the growth of mammalian cells, comprising approximately 10% of the total membrane of various mammalian tissues and cultured cells (Kuge et al. (1986) J. Biol. Chem. 261:5790-5794).
The enzyme responsible for the biosynthesis of PS in S. cerevisiae is CDPdiacylglycerol:L-serine O-phoshphatidyl transferase (Phosphatidylserine synthase; EC 2. 7. 8. 8). PS synthase catalyzes the formation of PS and CMP from CDP-diacylglycerol (CDP-DG) and serine by a sequential Bi Bi reaction mechanism (Bae-Lee et al. (1984) J. Biol. Chem. 259:10857-10862). PS synthase is an integral membrane protein. Detailed biochemical studies have shown that PS synthase activity is present in both the mitochondria and endoplasmic reticulum (Kuchler et al. (1986) J. Bacteriol. 165:901-910).
Phosphatidylserine (PS) synthase in Chinese Hamster Ovary cells (CHO) exists in two formsxe2x80x94(PSS) I and II. PSS I and PSS II are encoded by two genespssA andpssB, respectively (Kuge et al. (1997) J. Biol. Chem. 272: 19133-19139). PSS I is responsible for the conversion of phosphatidylcholine to phosphatidylserine and PSS II is responsible for the conversion of phosphatidylethanolamine to phosphatidylserine (Saito et al. (1998) J. Biol. Chem. 273:17199-17205). PS biosynthesis in CHO-K1 cells is inhibited upon the addition of PS to the culture medium suggesting that feedback control is involved in the regulation of PS biosynthesis (Nishijima et al. (1986) J. Biol. Chem. 261:5784-5789).
Various CHO mutants have been identified which exhibit defective synthesis in PSS I and PSS II. PSS I and PSS II are similar in sequence to each other; there is a 38% amino acid identity between the two PS synthases (Kuge et al. (1997) J Biol. Chem. 272:19133-19139). Results obtained by Kuge et al. indicate that PSS II in CHO-K1 cells is inhibited by exogenous PS and that the activity of over-produced PSS II in CHO-K1 cells is depressed for maintenance of the normal PS biosynthetic rate, probably through molecular mechanisms different from those for the exogenous PS-mediated inhibition. Also, the work of Kuge et al. demonstrated that the ARG-97 residue of PSS II is critical for both the exogenous PS-mediated inhibition of PS II and the depression of overproduced PSS II activity.
Because phospholipids such as PS are important components of eukaryotic membranes their proper biosynthesis is critical to cell homeostasis and function. Defects in PS synthase may yield membranes with altered functionality which could have implications in a wide range of disease states. Accordingly, PS synthases are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize novel PS synthases and tissues and disorders in which PS synthases are differentially expressed. The present invention advances the state of the art by providing novel human PS synthase molecules and the uses thereof.
Isolated nucleic acid molecules corresponding to human phosphatidylserine synthase-like nucleic acid sequences are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO:2. Further provided are human phosphatidylserine synthase-like polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein.
The present invention also provides vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as methods of making such vectors and host cells and for using them for production of the polypeptides or peptides of the invention by recombinant techniques.
The human phosphatidylserine synthase-like molecules of the present invention are useful for modulating the biosynthetic pathway involving the synthesis of the membrane phospholipid phosphatidylserine (PS). The molecules may be useful for a wide variety of human disorders as herein described.
Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding human phosphatidylserine synthase-like proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of human phosphatidylserine synthase-like encoding nucleic acids.
Another aspect of this invention features isolated or recombinant human phosphatidylserine synthase-like proteins and polypeptides. Preferred human phosphatidylserine synthase-like proteins and polypeptides possess at least one biological activity possessed by naturally occurring human phosphatidylserine synthase-like proteins.
Variant nucleic acid molecules and polypeptides substantially homologous to the nucleotide and amino acid sequences set forth in the sequence listings are encompassed by the present invention. Additionally, fragments and substantially homologous fragments of the nucleotide and amino acid sequences are provided.
Antibodies and antibody fragments that selectively bind the human phosphatidylserine synthase-like polypeptides and fragments are provided. Such antibodies are useful in detecting the human phosphatidylserine synthase-like polypeptides.
In another aspect, the present invention provides a method for detecting the presence of human phosphatidylserine synthase-like activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of human phosphatidylserine synthase-like activity such that the presence of human phosphatidylserine synthase-like activity is detected in the biological sample.
In yet another aspect, the invention provides a method for modulating human phosphatidylserine synthase-like activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) human phosphatidylserine synthase-like activity or expression such that human phosphatidylserine synthase-like activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to human phosphatidylserine synthase-like protein. In another embodiment, the agent modulates expression of human phosphatidylserine synthase-like protein by modulating transcription of a human phosphatidylserine synthase-like gene, splicing of a human phosphatidylserine synthase-like mRNA, or translation of a human phosphatidylserine synthase-like mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the human phosphatidylsenrne synthase-like mRNA or the human phosphatidylserine synthase-like gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant human phosphatidylserine synthase-like protein activity or nucleic acid expression by administering an agent that is a human phosphatidylserine synthase-like modulator to the subject. In one embodiment, the human phosphatidylserine synthase-like modulator is a human phosphatidylserine synthase-like protein. In another embodiment, the human phosphatidylserine synthase-like modulator is a human phosphatidylserine synthase-like nucleic acid molecule. In other embodiments, the human phosphatidylserine synthase-like modulator is a peptide, peptidomimetic, or other small molecule.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of the following: (1) aberrant modification or mutation of a gene encoding a human phosphatidylserine synthase-like protein; (2) misregulation of a gene encoding a human phosphatidylserine synthase-like protein; and (3) aberrant post-translational modification of a human phosphatidylserine synthase-like protein, wherein a wild-type form of the gene encodes a protein with a human phosphatidylserine synthase-like activity.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of a human phosphatidylserine synthase-like protein. In general, such methods entail measuring a biological activity of a human phosphatidylserine synthase-like protein in the presence and absence of a test compound and identifying those compounds that alter the activity of the human phosphatidylserine synthase-like protein.
The invention also features methods for identifying a compound that modulates the expression of human phosphatidylserine synthase-like genes by measuring the expression of the human phosphatidylserine synthase-like sequences in the presence and absence of the compound.
Other features and advantages of the invention will be apparent from the following detailed description and claims.